Phosphorylation and regulation of AKT/PKB by the rictor-mTOR complex
In certain aspects, the invention relates to methods for identifying compounds which modulate Akt activity mediated by the rictor-mTOR complex and methods for treating or preventing a disorder that is associated with aberrant Akt activity.
This application is a Continuation in Part of U.S. application Ser. No. 11/341,908, filed Jan. 27, 2006 which claims the benefit of priority of U.S. Provisional Application Nos. 60/648,636 filed Jan. 28, 2005 and 60/654,734 filed Feb. 18, 2005, and also claims the benefit of priority of U.S. Provisional Application No. 60/959,797, filed on Jul. 16, 2007. The teachings of the referenced Applications are incorporated herein by reference in their entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with government support under R01 AI47389 awarded by National Institutes of Health and under CA55164, CA16672, and CA49639 awarded by National Cancer Institute. The government has certain rights in the invention.
BACKGROUND OF THE INVENTIONDeregulation of Akt/Protein Kinase B (PKB) is implicated in the pathogenesis of many disorders including cancer and diabetes. Akt/PKB activation requires the phosphorylation of threonine 308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and serine 473 within the C-terminal hydrophobic motif by an unknown kinase.
The Akt/PKB kinase is a well-characterized effector of phosphoinositide 3-kinase (PI3K) and its deregulation plays important roles in the pathogenesis of human cancers. PI3K is necessary for the activation of Akt/PKB and current models suggest that phosphatidylinositol-3,4,5-triphosphates produced upon growth factor stimulation recruit Akt/PKB to the plasma membrane by binding to its N-terminal pleckstrin homology (PH) domain. At the membrane Akt/PKB is phosphorylated on two key residues: threonine 308 of the activation loop by PDK1 (D. R. Alessi et al., Curr Biol 7, 261 (1997); L. Stephens et al., Science 279, 710 (1998)), and serine 473 in the hydrophobic motif of the C-terminal tail by a kinase whose identity has been elusive. The role of S473 phosphorylation is controversial, but there is an emerging view that it precedes the phosphorylation of T308 and is important for the recognition and activation of Akt/PKB by PDK1 (M. P. Scheid et al., Mol Cell Biol 22, 6247 (2002); J. Yang et al., Mol Cell 9, 1227 (2002); D. R. Alessi et al., Embo J 15, 6541 (1996)).
BRIEF SUMMARY OF THE INVENTIONIn certain embodiments, the invention provides an isolated, purified or recombinant complex comprising an mTOR polypeptide, a rictor polypeptide, and an Akt polypeptide. Optionally, the subject complex further comprises a GβL polypeptide in addition to the mTOR polypeptide, the rictor polypeptide, and the Akt polypeptide. As described herein, the mTOR polypeptide, the rictor polypeptide, and the Akt polypeptide include the respective wildtype polypeptides, fragments and variants thereof. Preferably, such polypeptides are of eukaryotic origin, such as mammalian origin (e.g., mouse or human).
In certain embodiments, the invention provides a method for inhibiting Akt activity in a cell, comprising contacting the cell with a compound which inhibits function of a rictor-mTOR complex. For example, the compound may inhibit activity or expression of either rictor or mTOR, or both. Alternatively, the compound may inhibit interaction between rictor and mTOR, or interaction between Akt and the rictor-mTOR complex. In certain cases, the compound inhibits assembly of the rictor-mTOR complex. Optionally, the compound inhibits phosphorylation of Akt on S473 by the rictor-mTOR complex. Examples of such compounds include, but are not limited to a peptide, a phosphopeptide, a small organic molecule, an antibody, and a peptidomimetic. Optionally, the compounds include rapamycin and analogs of rapamycin (e.g., CCI-779, temsirolimus, and everolimus). Methods of measuring Akt activity are well known in the art, including measuring Akt phosphorylation, Akt kinase activity, and any Akt-mediated signaling (such as regulating cell proliferation, promoting cell survival, and regulating downstream targets such as FKHR). Preferably, the cell is a human cell. In certain cases, the cell is a cancer cell, such as a cancer cell which has no expression or reduced expression of PTEN. Optionally, the cancer cell is a hematological cancer cell.
In certain embodiments, the invention provides a method of treating or preventing a disorder that is associated with aberrant Akt activity in a subject, comprising administering to the subject an effective amount of a compound that inhibits function of a rictor-mTOR complex. For example, the disorders associated with aberrant Akt activity include cancer (e.g., a cancer characterized by no expression or reduced expression of PTEN) and diabetes. Preferably, the subject is a human. The compound may inhibit activity or expression of either rictor or mTOR, or both. Alternatively, the compound may inhibit interaction between rictor and mTOR, or interaction between Akt and the rictor-mTOR complex. In certain cases, the compound inhibits assembly of the rictor-mTOR complex. Optionally, the compound inhibits phosphorylation of Akt on S473 by the rictor-mTOR complex. Examples of such compounds include, but are not limited to a peptide, a phosphopeptide, a small organic molecule, an antibody, and a peptidomimetic. Optionally, the disorder is a hematological cancer selected from the group consisting of acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute nonlymphoblastic leukemia (ANLL), acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), multiple myeloma, myelodysplastic syndrome (MDS) such as refractory anemia with excessive blast (RAEB) and RAEB in transformation to leukemia (RAEB-T), and chronic myelo-monocytic leukemia (CMML).
In certain embodiments, the invention provides a method of identifying an antagonist of Akt kinase, comprising: a) contacting a test agent with an Akt polypeptide and a rictor-mTOR complex under conditions appropriate for phosphorylation of Akt by the rictor-mTOR complex; and b) assaying for phosphorylation of Akt by the rictor-mTOR complex in the presence of the test agent, as compared to phosphorylation of Akt by the rictor-mTOR complex in the absence of test agent. If the test agent decreases phosphorylation of Akt by the rictor-mTOR complex, the test agent is an antagonist of Akt kinase. Optionally, the method is conducted in the presence of rapamycin.
Similarly, in certain embodiments, the invention provides a method of identifying an agonist of Akt kinase, comprising: a) contacting a test agent with an Akt polypeptide and a rictor-mTOR complex under conditions appropriate for phosphorylation of Akt by the rictor-mTOR complex; and b) assaying for phosphorylation of Akt by the rictor-mTOR complex in the presence of the test agent, as compared to phosphorylation of Akt by the rictor-mTOR complex in the absence of test agent. If the test agent increases phosphorylation of Akt by the rictor-mTOR complex, the test agent is an agonist of Akt kinase. Optionally, the method is conducted in the presence of rapamycin.
In further embodiments, the invention provides a method of identifying an antitumor agent, comprising: a) contacting a test agent with an Akt polypeptide and a rictor-mTOR complex under conditions appropriate for phosphorylation of Akt by the rictor-mTOR complex; and b) assaying for phosphorylation of Akt by the rictor-mTOR complex in the presence of the test agent, as compared to phosphorylation of Akt by the rictor-mTOR complex in the absence of test agent. If the test agent decreases phosphorylation of Akt by the rictor-mTOR complex, the test agent is an antitumor agent. Optionally, the method is conducted in the presence of rapamycin.
In certain embodiments, the invention provides a method of assessing rapamycin-sensitivity of a cell, comprising: a) contacting a test cell with rapamycin or a rapamycin analog; and b) assaying for phosphorylation of Akt in the presence of rapamycin or the rapamycin analog, as compared to phosphorylation of Akt in the absence of rapamycin or the rapamycin analog. The test cell is sensitive to rapamycin if rapamycin or the rapamycin analog decreases phosphorylation of Akt. For example, the cell is a cancer cell. Optionally, the cell is a human cell.
In certain embodiments, the invention provides a method of assessing rapamycin-sensitivity of a cell, comprising: a) contacting a test cell with rapamycin or a rapamycin analog; and b) assaying for the amount of rictor-mTOR complex in the presence of rapamycin or the rapamycin analog, as compared to the amount of rictor-mTOR complex in the absence of rapamycin or the rapamycin analog. The test cell is sensitive to rapamycin if rapamycin or the rapamycin analog decreases the amount of rictor-mTOR complex. For example, the cell is a cancer cell. Optionally, the cell is a human cell.
In certain embodiments, the invention provides a method of identifying an agent that enhances rapamycin sensitivity of a cell, comprising: a) contacting a cell with rapamycin or a rapamycin analog; b) contacting a test agent with the cell; b) assaying for the amount of rictor-mTOR complex in the presence of the test agent, as compared to the amount of rictor-mTOR complex in the absence of test agent. The test agent enhances rapamycin sensitivity of the cell if the test agent decreases the amount of rictor-mTOR complex in the cell. For example, the cell is a cancer cell. Optionally, the cell is a human cell.
In certain embodiments, the invention provides a method of enhancing rapamycin sensitivity in a patient, comprising administering to a patient in need thereof a therapeutically effective amount of the agent identified by the present methods. In certain cases, the patient has cancer.
In certain embodiments, the invention provides a method of decreasing an unwanted side effect of rapamycin in a cell, comprising contacting a cell with an agent that enhances Akt activity. For example, the agent increases the amount of rictor-mTOR complex in the presence of rapamycin. To illustrate, the cell is an adipocyte and the unwanted side effect of rapamycin is lipolysis. Optionally, the cell is a human cell.
In certain embodiments, the invention provides a method of decreasing an unwanted side effect of rapamycin in a patient, comprising administering to a patient in need thereof a therapeutically effective amount of the agent that enhances Akt activity. For example, the agent increases the amount of rictor-mTOR complex in the presence of rapamycin. To illustrate, the unwanted side effect of rapamycin is hyperlipidemia. Optionally, the patient is a human.
The present invention is based, at least in part, on Applicants' discovery that the Target of Rapamycin (TOR) kinase and its associated protein rictor are necessary for S473 phosphorylation of Akt/PKB in eukaryotic cells (e.g., Drosophila and human cells) and that prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.
A reduction in rictor or mTOR expression inhibited Akt/PKB effectors and promoted apoptosis. The rictor-mTOR complex directly phosphorylated Akt/PKB on S473 in vitro and facilitated T308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation. The molecular identity of the S473 kinase (S473K), at times referred to as “PDK2” or the “hydrophobic motif (HM) kinase”, has been hotly debated for many years. Several candidate S473 kinases have been proposed, including PDK1 (A. Balendran et al., Curr Biol 9, 393 (1999)), Integrin-Linked Kinase (ILK) (S. Persad et al., J Biol Chem 276, 27462 (2001)), Akt/PKB itself (A. Toker, A. C. Newton, J Biol Chem 275, 8271 (2000)), and, most recently, DNA-PKcs (J. Feng et al., J Biol Chem 279, 41189 (2004)). Many lines of evidence argue that neither PDK1, ILK, nor Akt/PKB is the physiological S473 kinase (M. R. Williams et al., Curr Biol 10, 439 (2000); D. K. Lynch et al., Oncogene 18, 8024 (1999); M. M. Hill et al., J Biol Chem 276, 25643 (2001)) and for several reasons DNA-PKcs is also unlikely to have this function. There is no Drosophila orthologue of DNA-PKcs (A. S. Dore et al., DNA Repair (Amst) 3, 33 (2004)), and, thus, if DNA-PKcs is a physiological S473K in mammals, a distinct kinase must play that role in flies even though all other core components of the pathway (e.g. PI3K, Akt/PKB, PDK1, PTEN) are well conserved. Moreover, it has not been shown that DNA-PKcs phosphorylates full length Akt/PKB, and DNA-PKcs null mice (G. E. Taccioli et al., Immunity 9, 355 (1998)) do not suffer the growth retardation or insulin signaling defects associated with Akt1/PKB1 (H. Cho et al., J Biol Chem 276, 38349 (2001); W. S. Chen et al., Genes Dev 15, 2203 (2001)) or Akt2/PKB2 (H. Cho et al., Science 292, 1728 (2001)) null mice, respectively.
Mammalian TOR (mTOR) is a large protein kinase that exists in two distinct complexes within cells: one that contains mTOR, GβL and raptor (D.-H. Kim et al., Cell 110, 163 (2002); D.-H. Kim et al., Molecular Cell 11, 895 (2003); K. Hara et al., Cell 110, 177 (2002); R. Loewith et al., Mol Cell 10, 457 (2002)), and another mTOR, GβL and rictor (R. Loewith et al., Mol Cell 10, 457 (2002); D. D. Sarbassov et al., Curr Biol 14, 1296 (2004)). The raptor-containing complex is sensitive to the drug rapamycin and regulates cell growth, in part by phosphorylating the hydrophobic motif of S6K1 (P. E. Burnett et al., PNAS 95, 1432 (1998)), a member of the AGC family of kinases to which Akt/PKB belongs. The rictor-containing complex does not appear to be rapamycin sensitive and its cellular function is just beginning to be understood (D. D. Sarbassov et al., Curr Biol 14, 1296 (2004)).
The drug rapamycin has important uses in cardiology, oncology, and transplantation medicine but its clinically relevant molecular effects are not well understood. When bound to FKBP12, rapamycin interacts with and inhibits a multiprotein complex composed of mTOR, mLST8, and raptor (mTORC1). The distinct complex of mTOR, mLST8, and rictor (mTORC2) does not interact with FKBP12-rapamycin and is not thought to be sensitive to rapamycin treatment. mTORC2 phosphorylates and activates the Akt/PKB kinase, a key regulator of cell survival that is hyperactive in cells lacking the PTEN tumor suppressor.
In certain aspects, Applicants discovered that prolonged rapamycin treatment of mammalian cells suppresses the assembly of mTORC2. In many, but not all cell types, rapamycin decreases the levels of intact mTORC2 below those needed to maintain Akt/PKB phosphorylation. In such cells, rapamycin inhibits Akt/PKB signaling to the FKHR and AFX transcription factors and potentiates a pro-apoptotic stimulus. These effects are reversed by the expression of an Akt/PKB mutant with a phospho-mimetic residue at the mTORC2 phosphorylation site. In tumors expressing the Akt/PKB mutant the capacity of rapamycin to trigger apoptosis and decrease tumor size is also reduced. Thus, Applicants describe an unforeseen mechanism of action for rapamycin that is cell type dependent and provides a potential molecular explanation for some of the beneficial as well as undesirable clinical effects of the drug. Moreover, Applicants' work indicates that rapamycin, an already clinically approved drug, can be used as an inhibitor of Akt/PKB function in certain tumor cell types.
For example, Applicants investigated the molecular effects of mTOR inhibition by rapamycin analog CCI-779 in AML cells. Unexpectedly, CCI-779 blocked AKT activation via inhibition of mTORC2 formation, and resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-Cyclins in AML cells. Similar observations were made in samples from patients with hematological malignancies who were treated with the rapamycin analogs temsirolimus or everolimus. Applicants' data provides first evidence that rapamycin analogs inhibit AKT signaling in primary AML cells both in vitro and in vivo, and support the therapeutic potential of mTOR inhibition strategies in leukemias.
In certain embodiments, the invention provides an isolated, purified or recombinant complex comprising an mTOR polypeptide, a rictor polypeptide, and an Akt polypeptide. Optionally, the subject complex further comprises a GβL polypeptide in addition to the mTOR polypeptide, the rictor polypeptide, and the Akt polypeptide. As described herein, the mTOR polypeptide, the rictor polypeptide, and the Akt polypeptide include the respective wildtype polypeptides, fragments and variants thereof. Preferably, such polypeptides are of eukaryotic origin, such as mammalian origin (e.g., mouse or human).
In certain embodiments, the invention provides a method for inhibiting Akt activity in a cell, comprising contacting the cell with a compound which inhibits function of a rictor-mTOR complex. For example, the compound may inhibit activity or expression of either rictor or mTOR, or both. Alternatively, the compound may inhibit interaction between rictor and mTOR, or interaction between Akt and the rictor-mTOR complex. Optionally, the compound inhibits phosphorylation of Akt on S473 by the rictor-mTOR complex. Examples of such compounds include, but are not limited to a peptide, a phosphopeptide, a small organic molecule, an antibody, and a peptidomimetic. Methods of measuring Akt activity are well known in the art, including measuring Akt phosphorylation, Akt kinase activity, and any Akt-mediated signaling (such as regulating cell proliferation, promoting cell survival, and regulating downstream targets such as FKHR). Preferably, the cell is a human cell. In certain cases, the cell is a cancer cell, such as a cancer cell which has no expression or reduced expression of PTEN.
In certain embodiments, the invention provides a method of treating or preventing a disorder that is associated with aberrant Akt activity in a subject, comprising administering to the subject an effective amount of a compound that inhibits function of a rictor-mTOR complex. For example, the disorders associated with aberrant Akt activity include cancer (e.g., a cancer characterized by no expression or reduced expression of PTEN) and diabetes. Preferably, the subject is a human. The compound may inhibit activity or expression of either rictor or mTOR, or both. Alternatively, the compound may inhibit interaction between rictor and mTOR, or interaction between Akt and the rictor-mTOR complex. In certain cases, the compound inhibits assembly of the rictor-mTOR complex. Optionally, the compound inhibits phosphorylation of Akt on S473 by the rictor-mTOR complex. Examples of such compounds include, but are not limited to a peptide, a phosphopeptide, a small organic molecule, an antibody, and a peptidomimetic.
In certain embodiments, the invention provides a method of identifying an antagonist of Akt kinase, comprising: a) contacting a test agent with an Akt polypeptide and a rictor-mTOR complex under conditions appropriate for phosphorylation of Akt by the rictor-mTOR complex; and b) assaying for phosphorylation of Akt by the rictor-mTOR complex in the presence of the test agent, as compared to phosphorylation of Akt by the rictor-mTOR complex in the absence of test agent. If the test agent decreases phosphorylation of Akt by the rictor-mTOR complex, the test agent is an antagonist of Akt kinase. Optionally, the method is conducted in the presence of rapamycin.
Similarly, in certain embodiments, the invention provides a method of identifying an agonist of Akt kinase. Such method comprises: a) contacting a test agent with an Akt polypeptide and a rictor-mTOR complex under conditions appropriate for phosphorylation of Akt by the rictor-mTOR complex; and b) assaying for phosphorylation of Akt by the rictor-mTOR complex in the presence of the test agent, as compared to phosphorylation of Akt by the rictor-mTOR complex in the absence of test agent. If the test agent increases phosphorylation of Akt by the rictor-mTOR complex, the test agent is an agonist of Akt kinase. Optionally, the method is conducted in the presence of rapamycin.
In further embodiments, the invention provides a method of identifying an antitumor agent. Such method comprises: a) contacting a test agent with an Akt polypeptide and a rictor-mTOR complex under conditions appropriate for phosphorylation of Akt by the rictor-mTOR complex; and b) assaying for phosphorylation of Akt by the rictor-mTOR complex in the presence of the test agent, as compared to phosphorylation of Akt by the rictor-mTOR complex in the absence of test agent. If the test agent decreases phosphorylation of Akt by the rictor-mTOR complex, the test agent is an antitumor agent. Optionally, the method is conducted in the presence of rapamycin.
In certain aspects, the present invention provides assays for identifying therapeutic agents which either interfere with or promote function of the mTOR-rictor complex. In other aspects, the present invention provides assays for identifying therapeutic agents which modulate (inhibit or enhance) function of Akt. In certain embodiments, an assay of the invention comprises screening for activation of a kinase such as mTOR kinase or Akt. For example, mammalian cells such as HeLa cells are contacted with a compound, and then lysed. mTOR kinase or Akt kinase is then immunoprecipitated and assayed for its activation by methods well known in the art. Optionally, the assays are conducted in the presence of rapamycin.
In certain aspects, agents of the invention may be used to treat certain diseases such as cancer or diabetes, or a disease or condition that is responsive to modulation of the mTOR-rictor complex or Akt. For example, a screening assay of the invention may involve an assay designed to assess the anti-tumor activity of a test agent. The parameters detected in a screening assay may be compared to a suitable reference. A suitable reference may be an assay run previously, in parallel or later that omits the test agent. A suitable reference may also be an average of previous measurements in the absence of the test agent. In general, the components of a screening assay mixture may be added in any order consistent with the overall activity to be assessed, but certain variations may be preferred. Optionally, in a screening assay, the effect of a test agent may be assessed by, for example, assessing the effect of the test agent on kinetics, steady-state and/or endpoint of the reaction.
Certain embodiments of the invention relate to assays for identifying agents that bind to an mTOR, a rictor, a GβL, or an Akt polypeptide, or a particular domain thereof. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, and immunoassays for protein binding. The purified protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions and design of test agents. In one embodiment, an assay of the invention detects agents which inhibit interaction between mTOR and rictor. In another example, an assay of the invention detects agents which inhibit interaction between Akt and an mTOR-rictor complex. In certain specific embodiments, the screening methods are conducted in the presence of rapamycin.
In additional embodiments of the invention, assay formats include purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. For example, simple binding assays can be used to detect agents which bind to a rictor, mTOR, Akt, or GβL polypeptide. Such binding assays may also identify agents that act by disrupting the interaction among any two of these polypeptides. Agents to be tested can be produced, for example, by bacteria, yeast or other organisms (e.g., natural products), produced chemically (e.g., small molecules, including peptidomimetics), or produced recombinantly. In one embodiment, the test agent is a small organic molecule having a molecular weight of less than about 2,000 daltons. Assaying rictor-containing complexes (e.g., a complex comprising an mTOR protein and a rictor protein) in the presence and absence of a candidate inhibitor, can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes.
Certain embodiments of the invention relate to methods of identifying an agent that enhances rapamycin sensitivity of a cell (e.g., a cancer cell). For example, such methods comprise: a) contacting a cell with rapamycin or a rapamycin analog; b) contacting a test agent with the cell; b) assaying for the amount of rictor-mTOR complex in the presence of the test agent, as compared to the amount of rictor-mTOR complex in the absence of test agent. If the test agent decreases the amount of rictor-mTOR complex in the cell, the test agent is capable of enhancing rapamycin sensitivity of the cell. In other cases, the methods comprise: a) contacting a cell with rapamycin or a rapamycin analog; b) contacting a test agent with the cell; b) assaying for phosphorylation of Akt in the presence of rapamycin or the rapamycin analog, as compared to phosphorylation of Akt in the absence of rapamycin or the rapamycin analog. If the test agent decreases phosphorylation of Akt in the cell, the test agent is capable of enhancing rapamycin sensitivity of the cell. Optionally, the cell is a human cell.
Further embodiments of the invention relate to methods of assessing rapamycin-sensitivity of a cell (e.g., a cancer cell). For example, the methods comprise: a) contacting a test cell with rapamycin or a rapamycin analog; and b) assaying for the amount of rictor-mTOR complex in the presence of rapamycin or the rapamycin analog, as compared to the amount of rictor-mTOR complex in the absence of rapamycin or the rapamycin analog. The test cell is sensitive to rapamycin if rapamycin or the rapamycin analog decreases the amount of rictor-mTOR complex. In other cases, the methods comprise: a) contacting a test cell with rapamycin or a rapamycin analog; and b) assaying for phosphorylation of Akt in the presence of rapamycin or the rapamycin analog, as compared to phosphorylation of Akt in the absence of rapamycin or the rapamycin analog. The test cell is sensitive to rapamycin if rapamycin or the rapamycin analog decreases phosphorylation of Akt. Optionally, the cell is a human cell.
In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays of the present invention which are performed in cell-free systems, such as may be developed with purified or semi-purified proteins or with lysates, are often preferred as “primary” screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target.
In certain aspects, the present invention provides a method of treatment for a disease (disorder or condition) affected by aberrant activity of Akt or mTOR-rictor complex, by administering a compound that regulate activity of Akt or mTOR-rictor complex. Any disease that is responsive to Akt or mTOR-rictor complex modulation can be treated by the method of the invention. Examples of such diseases include, but are not limited to, cancer, diabetes, and cardiovascular diseases (e.g., restenosis).
Many diseases or conditions are characterized by or caused by aberrant activation of Akt in an animal. An example of a disease or condition is cancer. Particular examples of cancer include breast cancer, lung cancer, ovarian cancer, endometrial cancer, uterine cancer, brain cancer, sarcoma, melanoma, glioblastoma, leukemia, lymphoma, colorectal cancer, prostate cancer, pancreatic cancer, renal cell cancer, and liver cancer. Another disease or condition is rheumatologic disease, e.g., rheumatoid arthritis or osteoarthritis. A further example of the disease or condition is pulmonary disease, e.g., chronic obstructive pulmonary disease (COPD). The present invention further provides a method of increasing apoptosis of a cell (e.g., a cancer cell), comprising contacting or treating the cell with a compound that is identified by the methods if the present invention.
In certain specific embodiments, the present invention provides methods for treating hematological cancer including leukemia or lymphoma in a patient. Such methods comprise administering to said patient, to which such treatment is needed, an effective amount of a compound or pharmaceutical compositions containing the compound as disclosed in the application. Examples of hematological cancer that can be treated by the present methods include, but are not limited to, acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute nonlymphoblastic leukemia (ANLL), acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), multiple myeloma, myelodysplastic syndrome (MDS) such as refractory anemia with excessive blast (RAEB) and RAEB in transformation to leukemia (RAEB-T), and chronic myelo-monocytic leukemia (CMML). Optionally, the hematological cancer to be treated is acute promyelocytic leukemia (APL), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL) and multiple myeloma. Further examples of lymphoma that can be treated by the present methods include, but are not limited to, high grade lymphoma, intermediate grade lymphoma and low grade lymphoma. Preferably, the lymphoma to be treated is non-Hodgkin's lymphoma. The skilled artisan will recognize that other cancers, particularly hematological cancers, may be treated in accordance with the present invention.
In certain embodiments, the present invention provides combination or multiple therapies for a condition characterized by or caused by aberrant activation of Akt. For example, the subject methods and compounds may be used in combination with other therapeutic agents, including, but not limited to, anti-cancer agents, antiviral agents, and anti-diabetic agents.
In certain aspects, the present invention provides a method of enhancing rapamycin sensitivity in a patient (e.g., a cancer patient). For example, the method comprises administering to a patient in need thereof a therapeutically effective amount of an agent which decreases phosphorylation of Akt mediated by rictor:mTOR complex or an agent which decreases the amount of rictor:mTOR complex.
In certain aspects, the present invention provides a method of decreasing an unwanted side effect of rapamycin in a patient. For example, the method comprises administering to a patient in need thereof a therapeutically effective amount of the agent that enhances Akt activity in the presence of rapamycin or an agent that enhances assembly of the rictor:mTOR complex in the presence of rapamycin. An example of the unwanted side effect of rapamycin is hyperlipidemia in the patient. Preferably, the patient is a human. In certain specific aspects, the present invention provides a method of decreasing an unwanted side effect of rapamycin in a cell such as an adipocyte. For example, the method comprises contacting a cell with an agent that enhances Akt activity in the presence of rapamycin or an agent that enhances assembly of the rictor:mTOR complex in the presence of rapamycin. An example of the unwanted side effect of rapamycin is lipolysis. Preferably, the cell is a human cell.
When administered to an individual, the compounds of the invention can be administered as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.
A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the active therapeutic compound. The physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.
One skilled in the art would know that a pharmaceutical composition can be administered to a subject by various routes including, for example, oral administration; intramuscular administration; intravenous administration; anal administration; vaginal administration; parenteral administration; nasal administration; intraperitoneal administration; subcutaneous administration and topical administration. The composition can be administered by injection or by incubation. The pharmaceutical composition also can be linked to a liposome or other polymer matrix. Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
EXEMPLIFICATION Example 1 Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR ComplexApplicants used RNA interference (RNAi) in cultured Drosophila cells to determine the role of TOR pathway components in the phosphorylation of the hydrophobic motif sites of dAkt/dPKB and dS6K. In mammals and Drosophila, S6K suppresses signaling through the PI3K/Akt pathway so that inhibition of S6K boosts Akt/PKB phosphorylation (F. Tremblay, A. Marette, J Biol Chem 276, 38052 (2001); T. Radimerski et al., Genes Dev 16, 2627 (2002); L. S. Harrington et al., J Cell Biol 166, 213 (2004)). Knockdown of dS6K or dRaptor expression with double stranded RNAs (dsRNAs) inhibited the phosphorylation and activity of dS6K and increased the phosphorylation of dAkt/dPKB (
Applicants have shown that in Drosophila and human cells the Target of Rapamycin (TOR) kinase and its associated protein rictor are necessary for 5473 phosphorylation and that a reduction in rictor or mTOR expression inhibited an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on S473 in vitro and facilitated T308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation.
Because basal dAkt/dPKB phosphorylation is low in Drosophila Kc167 cells (
Results in Drosophila cells suggest that dTOR and dRictor have a shared positive role in the phosphorylation of the hydrophobic motif site of dAkt/dPKB. This finding was unexpected because previously (D.-H. Kim et al., Cell 110, 163 (2002)). Applicants observed no decrease in the phosphorylation of the hydrophobic motif site of Akt/PKB after reducing mTOR expression in human cells with small interfering RNAs (siRNAs). In retrospect, however, these experiments were undertaken when RNAi-mediated knockdowns of expression in mammalian cells were relatively inefficient. Here, using a lentiviral short hairpin RNA (shRNA) expression system that robustly suppresses gene expression (D. D. Sarbassov et al., Curr Biol 14, 1296 (2004)), Applicants obtained results in human cell lines analogous to those in Drosophila cells (
In a related study, it is shown that Rictor and mTOR, but not raptor, positively regulate the phosphorylation of serine 473 of Akt/PKB in a cell line that is null for DNA-PKcs (
As the rictor and mTOR knockdowns inhibit phosphorylation events critical for Akt/PKB activity, they should affect Akt/PKB-regulated processes. In HeLa cells, a reduction in the expression of rictor or mTOR, but not raptor, decreased phosphorylation of FKHR (Foxo 1) and AFX (Foxo4a) (
Because the raptor-mTOR complex directly phosphorylates the hydrophobic motif site of S6K1 (P. E. Burnett et al., PNAS 95, 1432 (1998)), Applicants determined if rictor-mTOR has an analogous function for Akt/PKB. Rictor-mTOR complexes isolated from HEK-293T or HeLa phosphorylated S473 but not T308 of full length, wild-type Akt/PKB in vitro (
To determine whether the phosphorylation of Akt/PKB on S473 by rictor-mTOR activates Akt/PKB activity, Applicants first used rictor-mTOR to phosphorylate Akt/PKB on S473 and then added PDK1 to the assay to phosphorylate T308. Prior phosphorylation of Akt/PKB on S473 boosted subsequent phosphorylation by PDK1 of T308 (
Results presented herein indicate that the rictor-mTOR complex is a hydrophobic motif kinase for Akt/PKB (
Reagents were obtained from the following sources: protein G-sepharose from Pierce; ATP-[γ-32P] from NEN; compounds LY294002, wortmannin, and staurosporine were obtained from Calbiochem; DMEM from Life Technologies; mTOR, S6K1, ATM, α-tubulin, and PKCα antibodies as well as HRP-labeled anti-mouse, anti-goat, and anti-rabbit secondary antibodies from Santa Cruz Biotechnology; phospho-T389 S6K1, phospho-S473 and phospho-T308 Akt/PKB, Akt/PKB, phospho-5256 FKHR (also recognizes phospho-S193 of AFX), FKHR, AFX, cleaved Caspase 3, phospho-S505 Drosophila Akt/PKB, and Drosophila Akt/PKB antibodies from Cell Signaling; Drosophila S6K antibody from Mary Stewart, North Dakota State University; and the rictor and raptor antibodies were previously described (D. D. Sarbassov et al., Curr Biol 14, 1296 (2004)). The Cell Death Detection Elisa Plus kit (Roche, #1774425) was used as described by the manufacturer to quantify DNA fragmentation during apoptosis. All cell lines were obtained from ATCC.
2. Cell LysisCells growing in 10 cm dishes were rinsed once with cold PBS and lysed on ice for 20 mM in 1 ml of ice-cold Lysis Buffer (40 mM Hepes pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate, and EDTA-free protease inhibitors (Roche)) containing 1% Triton X-100. After clearing of Triton X-100 material by centrifugation at 13,000×g for 10 min, samples containing 50-100 μg of protein were resolved by SDS-PAGE and proteins transferred to PVDF and visualized by immunoblotting as described (D.-H. Kim et al., Cell 110, 163 (2002)). For experiments with FKHR and AFX the Triton X-100 insoluble materials were solubilized in 1% SDS in 10 mM Tris-HCl pH 7.4 by heating at 100° C. for 3 minutes followed by a brief sonication. Equal protein amounts were then analyzed by immunoblotting.
3. Immunoprecipitations and Kinase AssaysFor all immunoprecipitation experiments the lysis buffer contained 0.3% CHAPS instead of 1% Triton in order to preserve the integrity of the mTOR complexes (D. D. Sarbassov et al., Curr Biol 14, 1296 (2004); D.-H. Kim et al., Cell 110, 163 (2002)). 4 μg of the indicated antibodies were added to the cleared cellular lysates and incubated with rotation for 90-min. 25 μl of a 50% slurry of protein G-sepharose was then added and the incubation continued for 1 h. Immunoprecipitates captured with protein G-sepharose were washed four times with the CHAPS Lysis Buffer and once with the rictor-mTOR kinase buffer (25 mM Hepes pH 7.5, 100 mM potassium acetate, 1 mM MgCl2). For kinase reaction immunoprecipitates were incubated in a final volume of 15 μl for 20 min at 37° C. in the rictor-mTOR kinase buffer containing 500 ng inactive Akt1/PKB1 (Ak1t/PKB1, Upstate Biotechnology, #14-279) and 500 μM ATP. The reaction was stopped by the addition of 200 μl ice-cold Enzyme Dilution buffer (20 mM MOPS, pH 7.0, 1 mM EDTA, 0.01% Brij 35, 5% glycerol, 0.1% 2-mercaptoethanol, 1 mg/ml BSA). After a quick spin, the supernatant was removed from the protein G-sepharose and analyzed by immunoblotting (D.-H. Kim et al., Cell 110, 163 (2002)). For experiments involving PDK1, the rictor-mTOR phosphorylation was performed as described above and the second reaction was initiated by adding to the samples 100 ng of PDK1 (Upstate Biotechnology, #14-452) and 5 μl of Mg/ATP Cocktail (220 mM MOPS, pH-7.2, 75 mM MgCl2, 500 μM ATP, 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT; Upstate Biotechnology, #20-113). The samples were incubated for a further 20 min at 37° C., the reactions stopped by adding 40 μl Enzyme Dilution buffer and the samples quickly spun to pellet the protein G-sepharose. Supernatants were used in the Akt1/PKB1 kinase assay as described below and were also analyzed by immunoblotting. The pelleted G-sepharose beads were also analyzed by immunoblotting to determine the levels of rictor, mTOR, and raptor in the immunoprecipitates. Akt1/PKB1 kinase activity was determined using Crosstide (Upstate Biotechnology, #12-331) as substrate as recommended by the manufacturers protocol. Briefly, supernatant samples containing phosphorylated Akt1/PKB1 were incubated for 10 min at 30° C. in a final volume of 25 μl of Akt/PKB kinase buffer (8 mM MOPS pH 7.0, 0.2 mM EDTA) containing 2.5 μl of Crosstide peptide (30 μM final concentration), 4.5 μl of Mg/ATP Cocktail, and 10 μl of [γ-32]ATP. After the incubation the samples were cooled on ice and 20 μl aliquots were transferred onto the center of P81 paper square (Upstate Biotechnology, #20-134). After drying the P81 paper squares were washed 3 times for 5 min each time with 0.75% phosphoric acid and once for 5 min with acetone. After the washing, the P81 squares were dried and radioactivity read in a scintillation counter.
4. Drosophila RNAi and AnalysisdsRNAs targeting Drosophila TOR pathway components were synthesized by in vitro transcription in 20 μl reactions using a T7 MEGAscript™ kit (Ambion). DNA templates for IVT were generated by RT-PCR from total Drosophila cellular RNA using the OneStep RT-PCR kit (Qiagen). Primers (which incorporated a 5′ and 3′ T7 promoter) for dAkt and dPTEN dsRNA synthesis were as follows:
The underlined region indicates the T7 promoter sequence. Primers for the synthesis of other dsRNAs were previously described (D. D. Sarbassov et al., Curr Biol 14, 1296 (2004)). dsRNA products were purified by adding 80 μl of RNAse free water to IVT reactions and filter purified with a vacuum manifold using Millipore filter plates (MANU 030 PCR). Final dsRNA concentrations were measured on a Nano-drop spectrophotometer.
Drosophila Kc167 cells were prepared for dsRNA addition by diluting an overnight culture seeded at 80×106 total cells in 12 ml Drosophila Schneider's medium to 1×106 cells/ml in Schneider's. 2 ml of media containing cells was then seeded to each well in 6-well culture dishes. dsRNAs were administered to cells using FuGENE 6 transfection reagent (Roche). Briefly, 3 μl of FuGENE was added to 97 μl of Drosophila SFM (Invitrogen), followed by addition of 2 μg of the indicated dsRNA in a sterile eppendorf tube. Tubes were gently mixed and incubated for 15 minutes at room temperature. FuGENE:dsRNA complexes were then administered to cells by adding the entire mix drop-wise around wells and then swirling to ensure even dispersal. For combination dsRNA addition experiments, 1.0 μg of PTEN dsRNA was mixed with 1.0 μg of the indicated dsRNA species (except in the GFP only control which contained 2.0 μg of the GFP dsRNA). Additional FuGENE:dsRNA complexes were added to wells on each of the following 2 days. On the third day of dsRNA addition, the medium was changed to avoid potential negative effects of excessive FuGENE on cell viability. After 4 days total of incubation to allow turnover of the target mRNAs, cell lysates were prepared as described (D. D. Sarbassov et al., Curr Biol 14, 1296 (2004)). 50 μg of total cellular protein was loaded per lane on 8% SDS-PAGE gels, separated, transferred to nitrocellulose membranes and analyzed by immunoblotting.
5. Lentiviral shRNA Cloning, Production, and Infection
Desalted oligonucleotides (IDT) were cloned into LKO.1 (S. A. Stewart et al., RNA 9, 493 (2003)) with the Age I/EcoRI sites at the 3′ end of the human U6 promoter. The sequences of the oligonucleotides are as follows:
Plasmids were propagated in and purified from Stbl2 bacterial cells (Invitrogen) and co-transfected together with the Delta VPR and CMV VSVG plasmids into actively growing HEK-293T using FuGENE (Roche) as described (D. D. Sarbassov et al., Curr Biol 14, 1296 (2004); S. A. Stewart et al., RNA 9, 493 (2003)). Virus-containing supernatants were collected at 36 and 60 hours after transfection, and concentrated by ultracentrifugation for 1.5 hrs at 23,000 RPM in an SW28 rotor at 4° C. Pellets were resuspended overnight at 4° C. in 1/600th of the original volume. Cells were infected twice in the presence of 6 μg/ml protamine sulfate, selected for puromycin resistance and analyzed on the 7th day after infection. In previous work, Applicants noted that an acute knockdown of mTOR expression in HEK-293T cells using siRNAs also partially decreased raptor expression (D.-H. Kim et al., Cell 110, 163 (2002)). This effect is decreased in magnitude in the chronic mTOR knockdown cell lines made with lentivirally-expressed shRNAs.
Example 2 Prolonged Rapamycin Treatment Inhibits mTORC2 Assembly and Akt/PKBThe mammalian TOR (mTOR) protein nucleates two distinct multiprotein complexes that regulate different pathways (reviewed in Guertin, D. A. & Sabatini, D. M. Trends Mol Med 11, 353-61 (2005)). The mTOR complex 1 (mTORC1) consists of mTOR, raptor, and mLST8 (also known as GβL) and regulates cell growth through effectors such as S6K1. The mTOR complex 2 (mTORC2) contains mTOR, rictor, and mLST8 and recent work shows that it regulates Akt/PKB by phosphorylating it on S473 (Sarbassov, D. D. et al. Science 307, 1098-101 (2005); Hresko, R. C. & Mueckler, M. J Biol Chem (2005)). Together with the phosphorylation of T308 by PDK1, S473 phosphorylation is necessary for full Akt/PKB activation (Alessi, D. R. et al. Embo J 15, 6541-51 (1996)). FKBP12-rapamycin binds only to mTORC1, leading to the assumption that the drug exerts its clinical effects by specifically perturbing this complex and its downstream signaling pathway. Although FKBP12-rapamycin cannot bind to preformed mTORC2 (Sarbassov, D. D. et al. Curr Biol 14, 1296-302 (2004); Jacinto, E. et al. Nat Cell Biol 6, 1122-8 (2004)), it does bind to free mTOR (Brown, E. J. et al. Nature 369, 756-758 (1994); Sabatini, D. M. et al. Cell 78, 35-43 (1994); Sabers, C. J. et al. J. Biol. Chem. 270, 815-822 (1995)). Because mTOR molecules should be free when newly synthesized and when mTOR complexes turn over, long term exposure of cells to rapamycin should lead to the binding of FKBP12-rapamycin to a large fraction of the mTOR molecules within cells. As the binding of FKBP12-rapamycin to free mTOR may prevent the subsequent binding of rictor, Applicants hypothesized (Sarbassov, D. D. et al. Science 307, 1098-101 (2005)) that prolonged rapamycin treatment may inhibit Akt/PKB signaling by interfering with the assembly of mTORC2.
To determine if rapamycin can alter the levels of intact mTORC2, Applicants treated HeLa or PC3 cells with 100 nM rapamycin for 0.5, 1, 2, or 24 hours and compared the amounts of rictor and raptor bound to mTOR. Rapamycin had little effect on the expression levels of mTOR, raptor, or rictor, but, as expected (Kim, D.-H. et al. Cell 110, 163-175 (2002)), it strongly reduced the amounts of raptor recovered with mTOR within 30 minutes of addition to HeLa or PC3 cells (
Using a cross-linking assay Applicants previously demonstrated that the binding of FKBP12-rapamycin to mTORC1 does not break the raptor-mTOR interaction within cells but only weakens it so that it cannot survive biochemical isolation (Kim, D.-H. et al. Cell 110, 163-175 (2002)). A similar mechanism cannot explain the loss of the rictor-mTOR interaction in rapamycin-treated cells because FKBP12-rapamycin cannot bind to a formed mTORC2 (Sarbassov, D. D. et al. Curr Biol 14, 1296-302 (2004); Jacinto, E. et al. Nat Cell Biol 6, 1122-8 (2004)). Instead, Applicants suspected that after prolonged rapamycin treatment a large fraction of the rictor and mTOR molecules within cells are no longer associated with each other. Applicants tested this possibility in a modified version of the experiment in
To test this Applicants pulsed-labeled HeLa and PC3 cells with 35S-methionine/cysteine in the presence or absence of rapamycin and followed the amount of newly-synthesized (i.e. 35S-labeled) mTOR bound to rictor during a chase period with unlabeled amino acids. In the absence of rapamycin and at all times during the chase period Applicants readily detected newly-synthesized mTOR bound to immunoprecipitated rictor in both HeLa and PC3 cells (
Because the interaction of mTOR with rictor is necessary for mTOR to phosphorylate S473 of Akt/PKB, Applicants asked if a 24-hour treatment with rapamycin inhibits Akt/PKB phosphorylation. In several cell lines Applicants compared the effects of rapamycin on the phosphorylation of S473 of Akt/PKB and of T389 of S6K1, a well-known mTORC1 phosphorylation site (Burnett, P. E. et al. PNAS 95, 1432-1437 (1998)) (
Why does rapamycin inhibit Akt/PKB phosphorylation in only certain cell types? The experiments in
To test this hypothesis Applicants asked if it is possible to confer rapamycin-sensitive Akt/PKB phosphorylation to a cell line by partially decreasing the expression of mTOR. A reduction in total mTOR should decrease the levels of mTORC2 in the cells so that rapamycin-mediated suppression of mTORC2 assembly will leave insufficient amounts of mTORC2 to mediate Akt/PKB phosphorylation. This is exactly what Applicants observe. A partial knockdown of mTOR in HEK-293T, HeLa, and H460 cells is sufficient to render Akt/PKB phosphorylation rapamycin-sensitive in these cell lines (
Because S473 phosphorylation is required for full Akt/PKB activation (reviewed in Scheid, M. P. & Woodgett, J. R. FEBS Lett 546, 108-12 (2003)), Applicants expected that inhibition of S473 phosphorylation by rapamycin to suppress Akt/PKB signaling. In PC3 cells rapamycin inhibited the phosphorylation of FKHR (Foxo 1) and AFX (Foxo4a) (
Rapamycin had analogous effects in tumor xenografts made from these cell lines in immunocompromised mice. In tumors derived from vector alone PC3 cells rapamycin strongly decreased the phosphorylations of S473 and T308 of Akt/PKB without affecting Akt/PKB expression (
Applicants' findings may be of value for determining which cancers or other diseases should be treated with rapamycin or its analogues (CCI 779, RAD001, AP23573). It will be important to identify biomarkers that can predict if Akt/PKB is sensitive to rapamycin in a particular cell type and to design dosing regimens that ensure Akt/PKB inhibition. Applicants suspect that Applicants' existing predictive test—the amount of rictor-mTOR complex remaining after rapamycin treatment—will be difficult to perform in a clinical setting. To obtain a biomarker it will be necessary to understand why in certain cell lines (e.g., HeLa) a significant fraction of mTORC2 is able to assemble even in the presence of rapamycin while in other cell lines this does not happen. A possible mechanism is that in certain cell types a fraction of the mTORC2s assembles in such a way that the FKBP12-rapamycin binding site is never accessible to the drug, perhaps because an unidentified protein or post-translational modification blocks the binding site. Applicants' preliminary analyses have failed to find a strong correlation between the rapamycin-sensitivity of Akt/PKB phosphorylation in a cell line and the expression levels of rictor, mTOR, raptor, Akt/PKB, S6K1, or FKBP12; the rates of cell proliferation in culture; the concentration of rapamycin used; or the tissue of origin. In addition, the forced overexpression of FKBP12 does not affect the rapamycin sensitivity of Akt/PKB phosphorylation (
Applicants' work indicates that rapamycin inhibits Akt/PKB signaling in cells where the drug decreases the levels of intact mTORC2 below those needed to maintain the phosphorylation of S473 of Akt/PKB. Applicants suggest that rapamycin is a cell-type dependent inhibitor of mTORC2 function as well as a universal inhibitor of the mTORC1 pathway. Rapamycin is in clinical trials as a treatment for cancer and has established uses in preventing vascular restenosis and the immune rejection of transplanted organs. It is interesting to note that Akt/PKB has important roles in the pathological processes implicated in all these conditions. A high fraction of tumors have activated Akt/PKB signaling as a result of PTEN loss and these cancers may be particularly sensitive to rapamycin (reviewed in Guertin, D. A. & Sabatini, D. M. Trends Mol Med 11, 353-61 (2005)). Rapamycin is known to have anti-angiogenic effects (Guba, M. et al. Nat Med 8, 128-35 (2002)) and Applicants find that the drug strongly inhibits Akt/PKB in endothelial cells (
Reagents were obtained from the following sources: protein G-sepharose and Dithiobis[succinimidyl propionate] (DSP) from Pierce; rapamycin from Calbiochem; DMEM, RPMI, F12, and MCDB 131 from Life Technologies; Fetal Bovine Serum (FBS), Heat Inactivated Fetal Bovine Serum (IFS), and indole-3-carbinol (I3C) from Sigma; EGM-2 media from Cambrex; antibodies to mTOR, S6K1, and ATM as well as HRP-labeled anti-mouse, anti-goat, and anti-rabbit secondary antibodies from Santa Cruz Biotechnology; and antibodies to phospho-T389 S6K1, phospho-S6, phospho-S473 and phospho-T308 Akt/PKB, Akt/PKB (all three Akt/PKB-directed antibodies recognize the three known Akt/PKB isoforms), phospho-S256 FKHR (also recognizes phospho-S193 of AFX), and AFX from Cell Signaling Technologies. Antibodies to rictor and raptor were previously described (Sarbassov, D. D. et al. Curr Biol 14, 1296-302 (2004)).
2. Cell Lines.Cell lines were cultured in the following media: Jurkat, BJAB, SKW3, U937, Ishikawa, HepG2, A375, A549, and H460 cells in RPMI with 10% IFS; OPM2, Δ47, LNCaP, UACC-903, Kym-1, Rd88SC.10, rh30, and rSMC cells in RMPI with 10% FBS; PC3, HeLa, HeLa S3, U2OS, Mel-STR, u87, 786-0, HEK-293T, MD-MBA-231, MD-MBA-468, HT29, c2c12 and MEFs (p53−/−) cells in DMEM with 10% IFS; CACO2, 827, and SW480 cells in DMEM with 10% FBS; BJ fibroblasts in DMEM/F12 with 10% IFS; HUVECs in MCDB 131 media supplemented with EGM-2 and 5% FBS; and HMLE cells in 1:1 DMEM/F12 supplemented with insulin, epidermal growth factor (EGF), and hydrocortisone. All the above cell lines were cultured at a density that allowed cell division throughout the course of the experiment. 3T3-L1 cells were cultured and differentiated as described (Frost, S. C. & Lane, M. D. J Biol Chem 260, 2646-52 (1985)). Parental, vector control, and PTEN-null DLD1 cells were cultured as described (Lee, C. et al. Cancer Res 64, 6906-14 (2004)) as were Jurkat cells having a doxycycline-inducible PTEN (Xu, Z. et al. Cell Growth Differ 13, 285-96 (2002)).
3. Rapamycin-Treatment of Mice and Organ Harvest.1 mg of rapamycin was dissolved in 20 μl of ethanol, which was then diluted with Ringer's saline solution to a final concentration of 1 mg/ml directly before use. Three-month old male C57BL/6NTac (Taconic) mice were administered daily intraperitioneal injections of 10 mg/kg rapamycin or the drug vehicle for 7 days. Mice were then euthanized with CO2, organs were harvested into RIPA buffer, and homogenized with mechanical disruption followed by sonication. Lysates from vehicle- and rapamycin-treated organ pairs were normalized for protein content and analyzed by immunoblotting as described (Kim, D.-H. et al. Cell 110, 163-175 (2002)). The vehicle- and rapamycin-treated mice ate similar amounts during the 7-day treatment period and at necropsy all mice had evidence of processed food in their stomachs and small intestines. Control experiments using phospho-S6 as a marker of the effectiveness of rapamycin reveals that the drug penetrates all major tissues except the brain. The experiment was repeated twice with similar results.
4. Cell Lysis, Immunoblotting, and Cross-Linking Assay.Cells growing in 10 cm diameter dishes were rinsed once with cold PBS and lysed on ice for 20 min in 1 ml of ice-cold Buffer A (40 mM Hepes pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate, and EDTA-free protease inhibitors (Roche)) containing 1% Triton X-100. After clearing of the lysates by centrifugation at 13,000×g for 10 mM, samples containing 50-100 μg of protein were resolved by SDS-PAGE and proteins transferred to PVDF and visualized by immunoblotting as described (Kim, D.-H. et al. Cell 110, 163-175 (2002)). For experiments with FKHR and AFX the Triton X-100 insoluble materials were solubilized in 1% SDS in 10 mM Tris-HCl pH 7.4 by heating at 100° C. for 3 minutes followed by a brief sonication. Equal protein amounts were then analyzed by immunoblotting. For standard immunoprecipitation experiments the cell lysis buffer consisted of Buffer A containing 0.3% CHAPS instead of 1% Triton X-100 in order to preserve the integrity of the mTOR complexes (Sarbassov, D. D. et al. Curr Biol 14, 1296-302 (2004); Kim, D.-H. et al. Cell 110, 163-175 (2002)). When used, DSP was prepared as a stock solution of 50 mg in 200 μl of DMSO and added to a final concentration in the cell culture medium of 1 mg/ml (2.5 mM) (Sarbassov, D. D. et al. Curr Biol 14, 1296-302 (2004); Kim, D.-H. et al. Cell 110, 163-175 (2002)). Cells were then incubated at 37° C., 5% CO2 and after 10 minutes the DSP was quenched by adding Tris-HCL, pH 8.0 to a final concentration of 100 mM. After a further 10 minute incubation at 37° C., 5% CO2 cells were lysed in Buffer A containing Triton X-100. On occasion DSP used at these high concentrations can form a precipitate but this has no effect on the performance of the cross-linking assay. Reducing conditions were used during the SDS-PAGE analysis of immunoprecipitates prepared from DSP-treated cells to ensure breaking of the cross-linking disulfide bonds.
5. Immunoprecipitations and Kinase Assays.To the cleared lysates prepared as above 4 μg of mTOR, rictor, or ATM antibodies was added per 1.2 mg of soluble protein and immune complexes were allowed to form by incubating with rotation for 90 minutes at 4° C. 25 μl of a 50% slurry of protein G-sepharose was then added and the incubation continued for 1 h. Immunoprecipitates captured with protein G-sepharose were washed four times with CHAPS-containing Buffer A and analyzed by immunoblotting as described (Sarbassov, D. D. et al. Curr Biol 14, 1296-302 (2004)). Immunoprecipitates used in kinase assays were also washed once with the rictor-mTOR kinase buffer (25 mM Hepes pH 7.5, 100 mM potassium acetate, 1 mM MgCl2). In kinase reactions immunoprecipitates were incubated in a final volume of 15 μl for 20 min at 37° C. in the rictor-mTOR kinase buffer containing 500 ng inactive Akt1/PKB1 (Akt1/PKB1, Upstate Biotechnology, #14-279) and 500 μM ATP. The reaction was stopped by the addition of 200 μl ice-cold Enzyme Dilution buffer (20 mM MOPS, pH 7.0, 1 mM EDTA, 0.01% Brij 35, 5% glycerol, 0.1% 2-mercaptoethanol, 1 mg/ml BSA). After a quick spin, the supernatant was removed from the protein G-sepharose and a 20 μl portion was analyzed by immunoblotting (Kim, D.-H. et al. Cell 110, 163-175 (2002)).
6. 35S-Labeling and Pulse-Chase Experiments.4×106 Hela or PC3 cells growing in 100 mm dishes were treated with 100 nM rapamycin or vehicle control for 20 minutes, rinsed once in methionine- and cysteine-free DMEM, and then incubated in 3.5 ml of the same medium containing 10% dialyzed serum and 0.1 mCi/ml of 35S-methionine/35S-cysteine (Express Protein Labeling Mix, Perkin Elmer). After allowing the cells to label for 30 minutes, the cells were washed once in the normal culture medium and incubated in fresh medium for the periods of time indicated in the figures. Cells were then lysed in CHAPS lysis buffer and rictor and mTOR immunoprecipitates prepared as described above. Quantification was performed using images acquired with a phosphoimager.
7. Lentiviral shRNA Cloning, Production, and Infection.
Lentiviral shRNAs were generated and used as described (Sarbassov, D. D. et al. Science 307, 1098-101 (2005)).
8. Apoptosis Induction and Detection.Cell lines stably expressing wild-type or S473D human Akt1/PKB1 were generated by infecting PC3 cells with retroviruses made from the MSCV vector system (Clontech). cDNAs were cloned into pMSCV-hygro at the Xho1/EcoRI site and retroviruses were generated as described (Ali, S. M. & Sabatini, D. M. J Biol Chem 280, 19445-8 (2005)). Cells were selected for one week in 200 μg/ml hygromycin before use. 15,000 PC3 (MSCV controls, WT Akt1/PKB1, or 473D Akt1/PKB1) cells were seeded in the wells of a 96 well-plate and cultured overnight. The next day the cells were rinsed once in serum-free medium and then cultured for 48 hrs in serum-free medium containing either DMSO (the small molecule vehicle), 100 nM rapamycin, 100 μM indole-3-carbinol, or both rapamycin and indole-3-carbinol. When indole-3-carbinol was used, 0.002% BSA was added to the medium. After 48 hours in culture all cells (adherent and floating) were processed with the Cell Death Detection Elisa plus (Roche, cat#1774425) as described by the manufacturer.
9. Tumor Xenografts, Immunohistochemistry, and In Situ Apoptosis Assays.PC3 cell lines stably expressing wild-type or S473D human Akt1/PKB1 or the empty vector were xenografted into six-week old immunodeficient mice (Ncr nu/nu mice; Taconic). All animal studies were performed according to the official guidelines from the MIT Committee on Animal Care and the American Association of Laboratory Animal Care. 3×106 PC3 cells were injected subcutaneously in the upper flank region of mice that had been anaesthetized with isoflurane. Tumors were allowed to grow to at least 50 mm3 in size and then treated with rapamycin (10 mg/kg) for two days. Mice were then sacrificed, the tumors excised, and tumor volumes estimated with the formula: volume=(a2×b)/2, where a=short and b=long tumor lengths, respectively, in millimeters. Sections of paraffin-embedded tumors on slides were processed for immunohistochemistry using the following primary antibodies and dilutions: 1:50 Akt1 (2H10, Cell Signaling Technology), 1:50 phospho-S473 Akt (736E11, Cell Signaling Technology), and 1:100 phospho-T308 Akt (244F9, Cell Signaling Technology). Briefly, sections were dewaxed and incubated in 3% H2O2 for 10 min at room temperature to quench endogeneous peroxidases and then processed for antigen retrieval by incubating in 10 mM sodium citrate buffer (pH 6) for 10 min in a sub-boiling water bath in a microwave oven. The sections were then incubated in blocking solution (5% horse serum in 1×TBST buffer) for 30 min at room temperature, washed three times, and then incubated overnight at 4° C. with primary antibody diluted in blocking solution. The next day, sections were incubated with the biotinylated secondary antibody for 1 hr at room temperature, washed three times, incubated 30 minutes with streptavidin-HRP (DakoCytomation), rewashed, and developed with DAB reagents (DakoCytomation) for 5-20 min until staining appeared. The slides were counterstained with hematoxylin, dehydrated, and mounted with coverslips. All washes were for 5 min in 1×TBST wash buffer (1×TBS with 0.1% Tween 20). An in situ cell death detection kit (Roche) was used as described by the manufacturer to detect apoptotic cells in tumors. Percentages of apoptotic cells per high-power field were quantified in a blinded fashion.
Example 3 Rapamycin Analogs Reduce mTORC2 Signaling and Inhibit AKT Activation in AMLThe mammalian target of rapamycin (mTOR) pathway regulates cell growth, proliferation and survival (Wullschleger et al., Cell 124,471 (2006)). mTOR, the central component of this pathway, partitions between two scaffold proteins, raptor and rictor. Upon activation, the rapamycin-sensitive raptor/mTOR protein complex (mTORC1) increases mRNA translation via activation of p70S6-kinase and inhibition of eIF4E binding protein 4EPB1 (Hara et al., Cell 110, 177 (2002)). The rictor/mTOR protein complex (mTORC2) was discovered only recently, is thought to be rapamycin-insensitive, and phosphorylates AKT in the hydrophobic Ser473 site. It is therefore essential for AKT activity (Sarbassov et al., Science 307, 1098 (2005)). Despite activity in model systems, the clinical anti-tumor activity of rapamycin analogues in patients has been modest (Wullschleger et al., Cell 124,471 (2006); Panwalkar et al., Cancer 100, 1578 (2004)) and only a fraction of patients respond (reviewed in Thomas G V, Curr Opin Genet Dev. 16, 78 (2006)). This has been attributed to the unanticipated ability of rapamycin to increase AKT activity via release of feedback inhibition of growth signaling pathways, both in cell systems and in tumor biopsies from patients6. However, in certain cell types prolonged inhibition of mTOR by rapamycin may impair mTORC2 assembly and hence AKT activation7. In this study, Applicants investigated the molecular consequences of mTOR inhibition in leukemic cells, both in vitro and in a clinical trial in vivo. Applicants' results demonstrate that rapamycin analogues suppress assembly of mTORC2 complex, resulting in marked inhibition of AKT signaling. Applicants propose that rapamycin-induced functional blockade of AKT in leukemic cells may define a subset of hematological malignancies that are likely to respond favorably to mTOR inhibition, and that inhibition of AKT signaling may serve as a valuable biomarker of mTOR inhibition in vivo.
Applicants first investigated the effects of prolonged (24 hrs) CCI-779 treatment on mTOR/raptor and mTOR/rictor complexes in U937 cells by immunoprecipitation/immunoblotting. CCI-779, without affecting the expression levels of mTOR, raptor or rictor, interrupted the mTORC1 and mTORC2 complex formation at concentrations of 1.25 μg/ml and higher (
Further, in 8 primary AML samples treated with CCI-779 in vitro for 24 hours, mTORC1 and mTORC2 formation were decreased by approximately 80% and 50%, respectively, in all samples tested, without affecting the expression of mTOR, raptor and rictor (see examples in
To determine if the above observations are clinically relevant, Applicants obtained peripheral blood samples from patients with hematological malignancies treated on Phase I/II protocols of the rapamycin analogues temsirolimus (25 mg intravenously every week) and everolimus (continuously at 5 or 10 mg orally daily)9. The levels of Ser473 phosphorylated AKT decreased in 3/5 patient samples at 1 or 24 hour(s) of temsirolimus treatment, and in 6/8 patient samples treated with everolimus (
PTEN expression was detected by Western blot analysis using the PTEN antibody that detects only the endogenous level of unmutated PTEN (Cell Signaling, Cat No. #9552). The expression level of PTEN was quantitated by densitometry and presented as a ratio to GAPDH expression. Wild-type PTEN was detected in all samples studied consistent with previously published reports.14 PB, peripheral blood; BM, bone marrow. MDS, myelodysplastic syndrome; RAEB, refractory anemia with excess blasts; RARS, refractory anemia with ringed sideroblasts.
For quantitation of Western blots, densitometric analyses were performed by calculating normalized ratios of phospho- to total AKT levels. Relative changes in expression were then calculated by comparing normalized protein expression post- and prior everolimus administration. “−” denotes a<30% or no change, ↓ denotes ≧30% decrease, and ↑≧30% increase in normalized phospho-protein expression. Biological activity was defined as either >50% decrease in peripheral blood absolute blast count (for acute leukemia patients) or absolute lymphocyte count (for CLL) for >1 week duration, or sustained increase in platelet count for >3 weeks duration. AML, acute myeloid leukemia; ALL, acute lymphocytic leukemia; CLL, chronic lymphocytic leukemia; MDS, myelodysplastic syndrome; RAEB, refractory anemia with excess blasts.
Quantitative changes in Cyclin D1, Cyclin D2 and Glut-1 transcription before and after treatment with rapamycin analogs (CCI-779 or RAD001). The relative amount of the transcripts of interest relative to that of β-2-microglobulin (β2M) of each sample was calculated as ΔCt. Data represent mean±SEM of the ΔCt values from 9 patients' samples with decreased pAKT (6 samples from patients treated with everolimus (RAD001) and 3 from patients treated with temsirolimus (CCI-779)); and from 4 patients' samples with no change or increase in pAKT (2 from patients treated with everolimus and 2 from patients treated with temsirolimus). The averaged ΔCt values before and after treatment were then compared using Wilcoxon pairwise non parametric method.
In summary, Applicants' observations suggest that rapamycin analogues potently inhibited AKT activity in leukemic cells via suppression of mTORC2 assembly, in addition to its well-characterized ability to suppress the mTORC1 pathway. Further, the data reported here provide the first in vivo evidence that rapamycin analogs are capable of suppressing AKT signaling in patients treated with these inhibitors. Applicants propose that this unforeseen mechanism of action of these agents define hematological tumors as malignancies, which are likely to respond favorably to pharmacological inhibition of mTOR signaling, in contrast to certain solid tumors in which the negative feedback loop from p70S6K to AKT may instead exacerbate cancer progression. Since dysregulation of components of the PI3K/AKT/mTOR pathway is a common event in hematological malignancies12,13, inhibition of mTOR signaling by rapamycin-like molecules may be a useful therapeutic strategy. Based on the results presented in this report Applicants speculate that the ability of rapamycin to inhibit AKT and its downstream pro-survival pathways may serve as a valuable biomarker in elucidating biologically effective doses and schedules of mTOR inhibitors in leukemia and cancer patients.
Materials and MethodsAML cell lines were cultured under standard conditions8 with rapamycin derivatives CCI-779 and RAD001. Bone marrow or peripheral blood samples for the in vitro studies were obtained from patients with newly diagnosed or recurrent AML after informed consent. Peripheral blood samples were obtained from relapsed or refractory patients with hematological malignancies treated with CCI-779 (temsirolimus, Wyeth Pharmaceuticals) or RAD001 (everolimus, Novartis Pharmaceuticals)9 after obtaining written informed consent. Clinical characteristics of patients are summarized in Table 1. Expression of total and phosphorylated AKT (Ser473), p70SSK (Thr389), 4EBP1 (Thr70), FoxO1 (Ser 256), PTEN were detected by Western blot analysis as previously reported. mTOR was immunoprecipitated using a specific anti-mTOR antibody (Santa Cruz, Calif.) and protein-A/G agarose (Santa Cruz, Calif.). Immune complexes were washed with CHAPS buffer3 and analyzed by Western blot as described7.
Rapamycin analogue CCI-779 (temsirolimus) for in vitro study was kindly provided by Dr. Janet Dancey (CTEP, National Cancer Institute, Rockville, Md.), and RAD001 (everolimus) by Novartis Pharmaceuticals.
Quantitative real-time PCR was performed as follow. Total RNA was isolated by Trizol (Invitrogen), and cDNA was then synthesized using SuperScript III (Invitrogen). Real time PCR was carried out using an ABI Prism 7700 instrument. Primers and probes for detecting human Cyclin D1 (Hs00277039_ml), Cyclin D2 (Hs00277041_ml), Glut-1 (Hs00197884_ml), and human β-2-microglobulin were purchased from TaqMan Gene Expression Assays (Applied Biosystems). The abundance of Cyclin D1/D2 and Glut-1 transcript relative to that of β-2-microglobulin was calculated as follows: relative expression (RE)=100×2 exp [−ΔCt], where ΔCt is the mean Ct of the transcript of interest less the mean Ct of the transcript for β-2-microglobulin.
INCORPORATION BY REFERENCEAll publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
Claims
1. A method for inhibiting Akt activity in a cell, comprising contacting the cell with a compound which inhibits function of a rictor-mTOR complex.
2. The method of claim 1, wherein the compound inhibits activity or expression of rictor.
3. (canceled)
4. The method of claim 1, wherein the compound inhibits interaction between rictor and mTOR.
5. The method of claim 1, wherein the compound inhibits assembly of the rictor-mTOR complex.
6. The method of claim 1, wherein the compound inhibits phosphorylation of Akt on S473 by the rictor-mTOR complex.
7. The method of claim 1, wherein the compound is rapamycin or a rapamycin analog.
8-12. (canceled)
13. The method of claim 1, wherein the cell is a hematological cancer cell.
14. (canceled)
15. A method of treating or preventing a disorder that is associated with aberrant Akt activity in a subject, comprising administering to the subject an effective amount of a compound that inhibits function of a rictor-mTOR complex.
16. (canceled)
17. The method of claim 15, wherein the disorder is a hematological cancer.
18. The method of claim 17, wherein the hematological cancer is selected from the group consisting of acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL), chronic myelocytic leukemia (CML), and chronic lymphocytic leukemia (CLL).
19. The method of claim 17, wherein the hematological cancer is selected from the group consisting of high grade lymphoma, intermediate grade lymphoma, and low grade lymphoma.
20-22. (canceled)
23. The method of claim 15, wherein the compound is rapamycin or a rapamycin analog.
24-27. (canceled)
28. A method of assessing rapamycin-sensitivity of a cell, comprising:
- a) contacting a test cell with rapamycin or a rapamycin analog; and
- b) assaying for phosphorylation of Akt in the presence of rapamycin or the rapamycin analog, as compared to phosphorylation of Akt in the absence of rapamycin or the rapamycin analog,
- wherein the test cell is sensitive to rapamycin if rapamycin or the rapamycin analog decreases phosphorylation of Akt.
29. The method of claim 28, wherein the cell is a cancer cell.
30. The method of claim 28, wherein the cell is a hematological cancer cell.
31. (canceled)
32. A method of assessing rapamycin-sensitivity of a cell, comprising:
- a) contacting a test cell with rapamycin or a rapamycin analog; and
- b) assaying for the amount of rictor-mTOR complex in the presence of rapamycin or the rapamycin analog, as compared to the amount of rictor-mTOR complex in the absence of rapamycin or the rapamycin analog,
- wherein the test cell is sensitive to rapamycin if rapamycin or the rapamycin analog decreases the amount of rictor-mTOR complex.
33. The method of claim 32, wherein the cell is a cancer cell.
34. The method of claim 32, wherein the cell is a hematological cancer cell.
35. (canceled)
36. A method of identifying an agent that enhances rapamycin sensitivity of a cell, comprising:
- a) contacting a cell with rapamycin or a rapamycin analog;
- b) contacting a test agent with the cell;
- b) assaying for the amount of rictor-mTOR complex in the presence of the test agent, as compared to the amount of rictor-mTOR complex in the absence of test agent,
- wherein the test agent enhances rapamycin sensitivity of the cell if the test agent decreases the amount of rictor-mTOR complex in the cell.
37. The method of claim 36, wherein the cell is a cancer cell.
38-49. (canceled)
Type: Application
Filed: Jul 15, 2008
Publication Date: Jul 8, 2010
Inventors: David M. Sabatini (Cambridge, MA), Dos D. Sarbassov (Shrewsbury, MA), Michael Andreeff (Houston, TX), Marina Konopleva (Houston, TX), Zhihong Zeng (Houston, TX), Francis J. Giles (San Antonio, TX)
Application Number: 12/218,629
International Classification: A61K 31/436 (20060101); C12N 5/00 (20060101); C12Q 1/02 (20060101); A61P 35/00 (20060101);
